Aptamer based affinity capture methods for the selective enrichment of human immunoglobulin fc domains

ABSTRACT

A method of capturing human immunoglobulin Fc domains in a biofluid sample is provided. The method includes providing an affinity capture device. The affinity capture device includes a surface having an aptamer that is at least 80% identical to SEQ ID NO 1 immobilized onto the surface of the affinity capture device. The biofluid sample is diluted with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) a magnesium cation at a concentration between about 10 µM to about 20 mM; and (C) a total monovalent cation concentration from 0 to no greater than 100 mM. The human immunoglobulin Fc domains in the biofluid sample are adsorbed to the aptamer with the binding buffer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 16/913,805,filed Jun. 26, 2020 entitled “Aptamer Based Affinity Capture Methods forthe Selective Enrichment of Human Immunoglobulin Fc Domains”, whichclaims priority to and benefit of U.S. Provisional Application No.62/866,830 filed on Jun. 26, 2019 and entitled “Aptamer Based AffinityCapture Methods for the Selective Enrichment of Human Immunoglobulin FcDomains.” The contents of each are incorporated herein by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a ST.26 Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference herein in its entirety. Said XML copy, created on Jan. 6,2023, is named W-4126-US03CON.xml and is 24,576 bytes in size.

FIELD OF THE TECHNOLOGY

The present disclosure relates to methods and kits for using an aptamerfor the selective capture of human Fc domains found in immunoglobulinsor contained within a humanized monoclonal antibody. More specifically,the present technology relates to improved selectivity and binding ofaptamer ligands to more effectively capture human Fc domains found inimmunoglobulins.

BACKGROUND

Over the past couple of decades, the market of immunotherapy has grownexponentially. Until 2018, over 80 antibody therapeutics have beenapproved by the FDA, and hundreds of candidates are undergoing clinicaltrials. Human monoclonal antibodies (mAb) represent one of the mostpopular antibody therapeutic modalities and offer significantly reducedimmunogenicity risks. About 80% of the FDA approved antibody drugs areeither fully human or humanized antibodies with more than 90% humansequence origin. To keep up with this rapid growth in demand for humanantibody therapeutics, there is a requirement for high-throughput,selective purification of target proteins from cell culture and biofluidsamples.

SUMMARY

What is needed are methods and kits for using an aptamer for theselective capture of human Fc domains found in immunoglobulins orcontained within a humanized monoclonal antibody. Due to the rapidgrowth in demand for human antibody therapeutics, the methods of thepresent technology have high-throughput and selective purification oftarget proteins from cell culture and biofluid samples. The technologyis robust and facilitates human Fc capture, enabling detailed andaccurate analyses during pharmacokinetic, pharmacodynamics andbiotransformation studies.

It was surprisingly found that there is a competing effect betweenmonovalent and divalent cations in a binding buffer used to bind theaptamer to a substrate as well as the surprising finding that thebinding buffer was susceptible to different cations. These surprisingfindings resulted in a common buffer being excluded from the presentmethod, as described in detail below. One benefit of the technology isthat a complex matrix (e.g., biofludis) can be reduced in its monovalentcation concentration and the negative impact of endogenous monovalentions is minimized, which provides improved binding capacity of anaptamer without loss of selectivity.

In one aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 1 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 1 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 1:5′ terminus - G G rA rG rG [i2FU] rG C [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C - 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 2 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 2 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 2:5′ terminus - G G rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] [i2FC] G A A A rG rG rA rA [i2FC] [i2FU]C C - 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 3 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 3 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 3:5′ terminus - G G rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] C G A A A rG rG rA rA [i2FC] [i2FU] C C- 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 4 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 4 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 4:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(O Me) - 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.C(OMe), G(OMe) and A(OMe) represent 2′-methoxy substituted nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 5 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 5 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 5:5′ terminus - G G rA rG rG [i2FU] rG C U(OMe) C C G A A A rG rG A A [i2FC] [i2FU] C C -  3′terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.U(OMe) represent 2′-methoxy substituted nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 6 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 6 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 6:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] U(OMe) [i2FC] [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.U(OMe), C(OMe), G(OMe) and A(OMe) represent 2′-methoxy substitutednucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 7 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 7 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 7:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] C(OMe) [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.C(OMe), G(OMe) and A(OMe) represent 2′-methoxy substituted nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 8 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 8 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 8:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] C(OMe) G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe)- 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.C(OMe), G(OMe) and A(OMe) represent 2′-methoxy substituted nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 9 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 9 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 9:5′ terminus - G G rA rG [i2FG] [i2FU] rG C [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotide.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 10 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 10 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 10:5′ terminus - G G rA rG rG [i2FU] [i2FG] C [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 11 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 11 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 11:5′ terminus - G G rA rG rG [i2FU] rG rC [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 12 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 12 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 12:5′ terminus - G G rA rG rG [i2FU] rG C rU C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′ terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.

In another aspect, the technology relates to a method of capturing humanimmunoglobulin Fc domains in a biofluid sample. The method includesproviding an affinity capture device comprising a surface having anaptamer that is at least 80% identical to SEQ ID NO 13 immobilized ontothe surface of the affinity capture device. The method also includesdiluting the biofluid sample with a binding buffer. The binding bufferincludes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine(TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The human immunoglobulin Fc domains in the biofluid sampleare adsorbed to the aptamer with the binding buffer. SEQ ID NO 13 isshown below. The method can include one or more of the embodimentsdescribed herein.

SEQ ID NO 13:5′ terminus - G G rA rG rG [i2FU] rG C U(OMe) C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.U(OMe) represents 2′-methoxy substituted nucleotides.

In some embodiments, the aptamer is at least 85%, 90%, 95%, 98% or 99%identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO13). The aptamer can be, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO 1 (orSEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ IDNO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages canbe used to form a range, for example, the aptamer can be between 85% to99% or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3,SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ IDNO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO12, or SEQ ID NO 13). In some embodiments, the aptamer is a23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer havingan extended or truncated sequence of about 3 residues. The aptamer canbe a 23-nucleotide aptamer having an extended or truncated sequence ofabout 5 residues. The aptamer can be covalently immobilized ornon-covalently immobilized onto the surface of the affinity capturedevice.

In some embodiments, the binding buffer has a concentration ofmonovalent cations less than about 50 mM. The binding buffer can have aconcentration of monovalent cations less than about 30 mM. In someembodiments, the pH of the binding buffer is between about 5 and about9. The pH of the binding buffer can be between about 6 to about 8. ThepH of the binding buffer can be about 7.2. For example, the pH of thebinding buffer can be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, or 9.0. These pH values can be used to form a range, forexample, from about 7.0 to about 7.4 or from about 7.1, to about 7.3.

The biofluid sample can be diluted. For example, the biofluid sample canbe diluted by a factor of 2, 10, or 20. In some embodiments, thebiofluid sample is diluted by a factor of 1, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The dilution factors can beused to form a range, for example, from about 2 to about 20 or fromabout 2 to about 10, or from about 10 to about 20. In some embodiments,the biofluid sample is diluted to obtain a total monovalent cationconcentation of the biofluid sample of no greater than 100 mM, nogreater than 50 mM or no greater than 30 mM. The total monovalent cationconcentration of the diluted biofluid sample can be no greater than 100mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, or 10 mM.

In some embodiments, the method also includes eluting the adsorbed humanimmunoglobulin Fc domain from the immobilized aptamer using an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM.The ammonium is in the form of tetramethylammonium, triethylammonium,ammonium formate, or ammonium acetate. In some embodiments, the ammoniumconcentration is between about 50 mM to about 500 mM. The eluent canhave a pH between about 6.5 to about 8.0. The eluent can have a pH ofabout 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, or 8.0. These pH values can be used to form a range, forexample, from between about 7.0 to about 8.0 or from about 6.5 to about7.5.

In some embodiments, the method also includes washing the adsorbed humanimmunoglobulin Fc domains with the binding buffer. The method can alsoinclude washing the adsorbed human immunoglobulin Fc domains with abuffer comprising Ca⁺.

In some embodiments, the concentration of the magnesium cation isbetween about 50 µM to about 1 mM. The concentration of the magnesiumcation can be, for example, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300µM, 350 µM, 400 µM, 450 µM, 500 µM, 550 µM, 600 µM, 650 µM, 700 µM, 750µM, 800 µM, 850 µM, 900 µM, 950 µM, or 1000 µM. These concentrations canbe used to form a range, for example, between about 50 µM to about 500µM or from about 50 µM to about 200 µM.

In some embodiments, the method also includes analyzing the eluted humanimmunoglobulin Fc domain with a detector. The detector can be asandwiched enzyme linked immunosorbent assay or a mass spectrometer.

In some embodiments, the human immunoglobulin Fc domains are containedwithin a humanized monoclonal antibody. The method can also includepurifying the humanized monoclonal antibody.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 1 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 2 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 3 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 4 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 5 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 6 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 7 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 8 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 9 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 10 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 11 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 12 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In another aspect, the technology relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 13 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The method also includeseluting the adsorbed human immunoglobulin Fc domain from the immobilizedaptamer using an eluent having an ammonium concentration between about10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The method can include one or more of the embodiments describedherein.

In some embodiments, the aptamer is at least 85%, 90%, 95%, 98% or 99%identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO13). The aptamer can be, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO 1 (orSEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ IDNO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages canbe used to form a range, for example, the aptamer can be between 85% to99% or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3,SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ IDNO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO12, or SEQ ID NO 13). In some embodiments, the aptamer is a23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer havingan extended or truncated sequence of about 3 residues. The aptamer canbe a 23-nucleotide aptamer having an extended or truncated sequence ofabout 5 residues. The aptamer can be covalently immobilized ornon-covalently immobilized onto the surface of the affinity capturedevice.

In some embodiments, the ammonium concentration is between about 50 mMto about 500 mM. For example, the ammonium concentration can be about 50mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or500 mM. These values can be used to form a range, for example, frombetween about 200 mM to about 400 mM.

In some embodiments, the eluent has a pH between about 6.5 to about 8.0.For example, the eluent can have a pH of about 6.5, 6.6, 6.7, 6.8, 6.9,7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. These pHvalues can be used to form a range, for example, between about 7.0 to8.0 or between about 6.5 to about 7.0.

The method can also include analyzing the eluted human immunoglobulin Fcdomain with a detector. The detector can be a sandwiched enzyme linkedimmunosorbent assay or a mass spectrometer.

In some embodiments, the human immunoglobulin Fc domains are containedwithin a humanized monoclonal antibody. The method can also includepurifying the humanized monoclonal antibody.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 1 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 2 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 3 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10µ M to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 4 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 5 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 6 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 7 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 8 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 9 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 10 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µ M to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 11 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 12 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In another aspect, the technology features a kit. The kits includes anaffinity capture device having a surface having an aptamer that is atleast 80% identical to SEQ ID NO 13 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of a bindingbuffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane(Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include one or more of the embodimentsdescribed herein.

In some embodiments, the aptamer is at least 85%, 90%, 95%, 98% or 99%identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO13). The aptamer can be, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO 1 (orSEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ IDNO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages canbe used to form a range, for example, the aptamer can be between 85% to99% or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3,SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ IDNO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO12, or SEQ ID NO 13). In some embodiments, the aptamer is a23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer havingan extended or truncated sequence of about 3 residues. The aptamer canbe a 23-nucleotide aptamer having an extended or truncated sequence ofabout 5 residues. The aptamer can be covalently immobilized ornon-covalently immobilized onto the surface of the affinity capturedevice.

In some embodiments, the binding buffer has a concentration ofmonovalent cations less than about 50 mM. The binding buffer can have aconcentration of monovalent cations less than about 30 mM. In someembodiments, the pH of the binding buffer is between about 5 and about9. The pH of the binding buffer can be between about 6 to about 8. ThepH of the binding buffer can be about 7.2. For example, the pH of thebinding buffer can be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, or 9.0. These pH values can be used to form a range, forexample, from about 7.0 to about 7.4 or from about 7.1, to about 7.3.

In some embodiments, the concentration of the magnesium cation isbetween about 50 µM to about 1 mM. The concentration of the magnesiumcation can be, for example, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300µM, 350 µM, 400 µM, 450 µM, 500 µM, 550 µM, 600 µM, 650 µM, 700 µM, 750µM, 800 µM, 850 µM, 900 µM, 950 µM, or 1000 µM. These concentrations canbe used to form a range, for example, between about 50 µM to about 500µM or from about 50 µM to about 200 µM.

In some embodiments, the kit also includes a vial of an eluent having anammonium concentration between about 10 mM to about 1000 mM. Theammonium can be in the form of tetramethylammonium, triethylammonium,ammonium formate, or ammonium acetate.

In some embodiments, the kit also includes a vial of a buffer comprisingCa⁺. The buffer can be concentrated and the kit can also includeinstructions for diluting the concentrated buffer to a workingconcentration using water. For example, the working concentration of thebuffer can be between about 0.5 and 20 mM and the concentratedconcentration can be between about 20 to 500 mM.

In some embodiments, the vial of buffer comprises 10 mM Tris, 5 mM CaCl₂at a pH of 7.2.

In some embodiments, the kit also includes instructions for performingany one of the methods described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 1 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 2 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 3 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 4 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 5 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 6 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 7 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 8 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 9 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 10 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 11 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 12 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In another aspect, the technology relates to a kit. The kit includes anaffinity capture device including a surface having an aptamer that is atleast 80% identical to SEQ ID NO 13 immobilized onto the surface of theaffinity capture device. The kit also includes a vial of an eluenthaving an ammonium concentration between about 10 mM to about 1000 mM,wherein the ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The kit caninclude one or more the embodiments described herein.

In some embodiments, the aptamer is at least 85%, 90%, 95%, 98% or 99%identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO13). The aptamer can be, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO 1 (orSEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ IDNO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages canbe used to form a range, for example, the aptamer can be between 85% to99% or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3,SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ IDNO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO12, or SEQ ID NO 13). In some embodiments, the aptamer is a23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer havingan extended or truncated sequence of about 3 residues. The aptamer canbe a 23-nucleotide aptamer having an extended or truncated sequence ofabout 5 residues. The aptamer can be covalently immobilized ornon-covalently immobilized onto the surface of the affinity capturedevice.

In some embodiments, the ammonium concentration is between about 50 mMto about 500 mM. For example, the ammonium concentration can be about 50mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or500 mM. These values can be used to form a range, for example, frombetween about 200 mM to about 400 mM.

In some embodiments, the eluent has a pH between about 6.5 to about 8.0.For example, the eluent can have a pH of about 6.5, 6.6, 6.7, 6.8, 6.9,7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. These pHvalues can be used to form a range, for example, between about 7.0 to8.0 or between about 6.5 to about 7.0.

The kit can also include instructions for performing any one of themethods described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart of a method of capturing human immunoglobulin Fcdomains in a biofluid sample, according to an illustrative embodiment ofthe technology.

FIG. 2A is a chart showing the impact of divalent cations on hIgGrecovered in eluate for various binding buffers, according to anillustrative embodiment of the technology. FIG. 2A depicts an example ofthe impact of divalent cations in binding buffer. Recovery andbreakthrough for 5 µg of aptamer immobilized onto 40 µL of streptavidinresin when loaded with 100 µg of human IgG. Buffer A contains: 10 mMTris HCl, pH 7.2; Buffer B contains: 10 mM Tris HCl, 5 mM MgCl₂, pH 7.2;Buffer C contains: 10 mM Tris HCl, 5 mM CaCl₂; Buffer D contains: 10 mMTris HCl, 5 mM MnCl₂; Buffer E contains: 10 mM Tris HCl, 5 mM Znacetate. Buffer Nakamura et al. contains: 145 mM NaCl, 5.4 mM KCl, 1.8mM CaCl₂, 0.8 mM MgCl₂, 20 mM Tris HCl, pH 7.6. Experimental details canbe found in Example 1.

FIG. 2B is a chart showing the impact of divalent cations on hIgG inbreakthrough for various binding buffers, according to an illustrativeembodiment of the technology. FIG. 2B depicts an example of the impactof divalent cations in binding buffer. Recovery and breakthrough for 5µg of aptamer immobilized onto 40 µL of streptavidin resin when loadedwith 100 µg of human IgG. Buffer A contains: 10 mM Tris HCl, pH 7.2;Buffer B contains: 10 mM Tris HCl, 5 mM MgCl₂, pH 7.2; Buffer Ccontains: 10 mM Tris HCl, 5 mM CaCl₂; Buffer D contains: 10 mM Tris HCl,5 mM MnCl₂; Buffer E contains: 10 mM Tris HCl, 5 mM Zn acetate. BufferNakamura et al contains: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mMMgCl₂, 20 mM Tris HCl₂, pH 7.6. Experimental details can be found inExample 1.

FIG. 3A is a chart showing the impact of monovalent cations on hIgGrecovered in eluate for various binding buffers, according to anillustrative embodiment of the technology. FIG. 3A depicts an example ofthe impact of monovalent cations in binding buffer. Recovery andbreakthrough for 5 µg of aptamer immobilized onto 40 µL of streptavidinresin when loaded with 100 µg of human IgG. Buffer A contains: 10 mMTris HCl, 150 mM NaCl, pH 7.2; Buffer B contains: 10 mM Tris HCl, 150 mMNaCl, 5 mM KCl. pH 7.2; Buffer C contains: 10 mM Tris HCl, 150 mM NaCl,5 mM MgCl₂ pH 7.2; Buffer D contains: 10 mM Tris HCl, 150 mM NaCl, 5 mMCaCl₂, pH 7.2; Buffer E contains: 10 mM Tris HCl, 150 mM NaCl, 5 mMMnCl₂, pH 7.2; Buffer F contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM Znacetate pH 7.2. Buffer G contains: 10 mM Tris HCl, 5 mM MgCl₂, pH 7.2;Buffer H contains: 10 mM Tris HCl, 5 mM CaCl₂, pH 7.2; Buffer Icontains: 10 mM Tris HCl, 5 mM MnCl₂, pH 7.2; Buffer J contains: 10 mMTris HCl, 5 mM Zn acetate, pH 7.2. Experimental details can be found inExample 1.

FIG. 3B is a chart showing the impact of monovalent cations on hIgG inbreakthrough for various binding buffers, according to an illustrativeembodiment of the technology. FIG. 3B depicts an example of the impactof monovalent cations in binding buffer. Recovery and breakthrough for 5µg of aptamer immobilized onto 40 µL of streptavidin resin when loadedwith 100 µg of human IgG. Buffer A contains: 10 mM Tris HCl,150 mM NaCl,pH 7.2; Buffer B contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM KCl, pH7.2; Buffer C contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM MgCl₂, pH 7.2;Buffer D contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM CaCl₂, pH 7.2;Buffer E contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM MnCl₂, pH 7.2;Buffer F contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM Zn acetate, pH 7.2.Buffer G contains: 10 mM Tris HCl, 5 mM MgCl₂, pH 7.2; Buffer Hcontains: 10 mM Tris HCl, 5 mM CaCl₂, pH 7.2; Buffer I contains: 10 mMTris HCl, 5 mM MnCl₂, pH 7.2; Buffer J contains: 10 mM Tris HCl, 5 mM Znacetate, pH 7.2. Experimental details can be found in Example 1.

FIG. 4A is a chart showing the impact of pH buffer component (tris vs.phosphate) on hIgG recovered in eluate, according to an illustrativeembodiment of the technology. FIG. 4A depicts an example of the impactof pH buffer component: tris vs. phosphate. Recovery and breakthroughfor 5 µg of aptamer immobilized onto 40 µL of streptavidin resin whenloaded with 100 µg of human IgG. Buffer A contains: 20 mM SodiumPhosphate, pH 7.0; Buffer B contains: 20 mM Sodium Phosphate, 150 mMNaCl, pH 7.0; Buffer C contains: 20 mM Sodium Phosphate, 5 mM MgCl₂ pH7.0; Buffer D contains: 10 mM Tris HCl pH 7.2; Buffer E contains: 10 mMTris HCl, 150 mM NaCl, pH 7.2; Buffer F contains: 10 mM Tris HCl, 5 mMMgCl₂ pH 7.2. Experimental details can be found in Example 1.

FIG. 4B is a chart showing the impact of pH buffer component (tris vs.phosphate) on hIgG in breakthrough, according to an illustrativeembodiment of the technology. FIG. 4B depicts an example of the impactof pH buffer component: tris vs. phosphate. Recovery and breakthroughfor 5 µg of aptamer immobilized onto 40 µL of streptavidin resin whenloaded with 100 µg of human IgG. Buffer A contains: 20 mM SodiumPhosphate, pH 7.0; Buffer B contains: 20 mM Sodium Phosphate, 150 mMNaCl, pH 7.0; Buffer C contains: 20 mM Sodium Phosphate, 5 mM MgCl₂, pH7.0; Buffer D contains: 10 mM Tris HCl, pH 7.2; Buffer E contains: 10 mMTris HCl, 150 mM NaCl, pH 7.2; Buffer F contains: 10 mM Tris HCl, 5 mMMgCl₂, pH 7.2. Experimental details can be found in Example 1.

FIG. 5 is a chart showing a non-specific binding study with differentbinding buffers, according to an illustrative embodiment of thetechnology. FIG. 5 depicts an example of a non-specific binding studywith different binding buffer. Recovery and breakthrough for 5 µg ofaptamer immobilized onto 40 µL of streptavidin resin when loaded with100 µg of human IgG. Buffer A contains: 10 mM Tris HCl, 150 mM NaCl, 5mM MgCl₂, pH 7.2; Buffer B contains: 10 mM Tris HCl, 150 mM NaCl, 5 mMCaCl₂, pH 7.2; Buffer C contains: 10 mM Tris HCl, 150 mM NaCl, 5 mMMnCl₂, pH 7.2; Buffer D contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM Znacetate, pH 7.2. Buffer E contains: 10 mM Tris HCl, 5 mM MgCl₂, pH 7.2;Buffer F contains: 10 mM Tris HCl, 5 mM CaCl₂, pH 7.2; Buffer Gcontains: 10 mM Tris HCl, 5 mM CaCl₂, pH 7.2; Buffer H contains: 10 mMTris HCl, 5 mM Zn acetate, pH 7.2. Experimental details can be found inExample 2.

FIG. 6 is a chart showing the impact of magnesium concentration inbinding buffer and section of elution buffer, according to anillustrative embodiment of the technology. FIG. 6 depicts an example ofthe impact of Mg concentration in binding buffer and selection ofelution buffer. Recovery and breakthrough for 5 µg of aptamerimmobilized onto 40 µL of streptavidin resin when loaded with 100 µg ofhuman IgG. Binding Buffer contains 10 mM Tris HCl, pH 7.2 with MgCl₂,concentration range from 1 µM to 200 mM. Captured hIgG was eluted witheither 200 mM NH₄ Acetate or 200 mM EDTA. Experimental details can befound in Example 2.

FIG. 7 is graph showing the binding capacity of aptamer versus singledomain antibody V_(HH), according to an illustrative embodiment of thetechnology. FIG. 7 is an example of the binding capacity of aptamerversus single domain antibody V_(HH). Recovery and breakthrough for 40µL of streptavidin resin with vary amount of anti-human Fc ligand(aptamer or V_(HH)) immobilized when loaded with 500 µg of human IgG.Experimental details can be found in Example 3.

FIGS. 8A-8C depict an example of non-specific binding of aptamer againstendogenous protein from biofluids.

FIG. 8A is an LC-UV profile of plasma sample, according to anillustrative embodiment of the technology.

FIG. 8B is an LC-UV profile of aptamer flow through, according to anillustrative embodiment of the technology.

FIG. 8C is an LC-UV profile of eluate fractions, according to anillustrative embodiment of the technology.

FIGS. 8D-8E depict an example of non-specific binding of aptamer againstendogenous protein from biofluids. LC-UV profile of eluate fractionsrecovered from 40 µL of streptavidin resin with 5 µg, 12.5 µg and 25 µgaptamer immobilized. The loading and recovery of NIST mAb and plasmaprotein were calculated accordingly and summarized in table (8E).Experimental details can be found in Example 4.

FIG. 8D is an LCUV overlay of eluate fractions recovered from 40 µg ofstreptavidin resin with 5 µg, 12.5 µg and 25 µg aptamer immobilized,according to an illustrative embodiment of the technology.

FIG. 8E is a table showing the loading and recovery of NIST mAb andplasma protein that were calculated, according to an illustrativeembodiment of the technology.

FIG. 9A, FIG. 9B, and FIG. 9C are examples of a procedure to reducenon-specific binding of aptamer against endogenous protein from ratplasma. Eluate and washing fractions for 5 µg of aptamer immobilizedonto 40 µL of streptavidin resin when loaded with 25 µg of NIST mAb and20 µL of rat plasma.

FIG. 9A is an overlay of LC-UV profile of aptamer 2^(nd) washingfractions with washing buffer A through F, according to an illustrativeembodiment of the technology. FIG. 9A is an example of an overlay ofLC-UV profile of aptamer 2^(nd) washing fractions with washing buffer Athrough F (top to bottom). Buffer A contains: 10 mM Tris HCl, 0.5 mMMgCl₂, pH 7.2 (Binding buffer); Buffer B contains: 10 mM Tris HCl, 5 mMCaCl₂, pH 7.2; Buffer C contains: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂,0.8 mM MgCl₂, 20 mM Tris HCl, pH 7.6 (Nakamura et. al.); Buffer Dcontains: 20 mM phosphate, pH 7.0; Buffer E contains: 0.1 M glycine HClpH 2.7; Buffer F contains: 150 mM NaCl.

FIG. 9B is an overlay of LC-UV profile of aptamer eluate fractions after2^(nd) washing step with washing buffer A through F, according to anillustrative embodiment of the technology. FIG. 9B is an example of anoverlay of LC-UV profile of aptamer eluate fractions after 2^(nd)washing step with washing buffer A through F (top to bottom).Experimental details can be found in Example 4.

FIG. 9C is a chart showing the calculated recovery of NIST mAb andplasma protein in both 2^(nd) washing and eluate fractions with 2^(nd)washing buffer A through F, according to an illustrative embodiment ofthe technology. FIG. 9C is an example of a procedure to reducenon-specific binding of aptamer against endogenous protein from ratplasma. Eluate and washing fractions for 5 µg of aptamer immobilizedonto 40 µL of streptavidin resin when loaded with 25 µg of NIST mAb and20 µL of rat plasma. C. The calculated recovery of NIST mAb and plasmaprotein in both 2^(nd) washing and eluate fractions with 2^(nd) washingbuffer A through F (top to bottom). Buffer A contains: 10 mM Tris HCl,0.5 mM MgCl₂, pH 7.2 (Binding buffer); Buffer B contains: 10 mM TrisHCl, 5 mM CaCl₂, pH 7.2; Buffer C contains: 145 mM NaCl, 5.4 mM KCI, 1.8mM CaCl₂, 0.8 mM MgCl₂, 20 mM Tris HCl, pH 7.6 (Nakamura et. al.);Buffer D contains: 20 mM phosphate, pH 7.0; Buffer E contains: 0.1 Mglycine HCl, pH 2.7; Buffer F contains: 150 mM NaCl. Experimentaldetails can be found in Example 4.

DETAILED DESCRIPTION

The technology includes methods and kits for using an aptamer for theselective capture of human Fc domains found in immunoglobulins. Withthis technology, the selectivity and binding capacity of certain aptamerligands have been significantly improved, making it more effective it isapplicability to the capture of human IgG (hIgG). Together withparticular embodiments of aptamer immobilized resins and form factorsfor affinity capture devices, a powerful and robust technology foranti-human Fc capture has been devised. In one exemplary embodiment, andan optimized workflow, an immobilized aptamer resin is contained withina pipet tip and used for the rapid and selective capture of thehumanized Fc domain contained within a monoclonal antibody (mAb)therapeutic from a sample of biofluid obtained during preclinical rodentor monkey trials.

Affinity capture approaches are some of the most powerful techniques forfacilitating antibody purification, antibody characterization, and thedevelopment of clinical diagnostics. The enrichment of target antibodiescan be simplified to a one-step process if appropriately designedaffinity consumables are available. Aptamers, a group of short,single-stranded oligonucleotides with unique 3D conformation have beenproposed as novel affinity ligands for high capacity and highselectivity enrichment. These chemically synthesized molecules areusually less than 10 kDa in size and their high affinity capability canbe achieved by targeted screening via SELEX (Systematic Evolution ofLigands by Exponential Enrichment) amplification. Because of theircomparatively small size, aptamers can achieve high surface coverage onsolid supports through controllable immobilization, minimize anypossible steric hindrance, and ensure the high yield of target proteinwith significantly reduced sample preparation time. Besides, being thatthey are in vitro synthesized oligonucleotides, aptamers are oftenhighly stable and they can be manufactured efficiently and economically,with potentially better batch-to-batch reproducibility versusprotein-based ligands.

Anti-human Fc aptamer ligands comprised of 40 or fewer nucleotides areshown in U. S. Pat. No. 8,637,656 to Nakamura et al. (incorporated byreference herein in its entirety) as protein A alternatives for thepurification of antibody and biotherapeutics. Previous studies involvingthese aptamers have shown that a GGUGCU bulging motif is essential forthe selective interaction with human IgG Fc domain. Two stem structuresat both ends of the bulge motif, together with a 3 or 4 nucleotide loopform the critical affinity motif of the molecule. Incorporation of2′-fluoro groups within select pyrimidine nucleotide residues was alsoapplied to improve the stability and selectivity of the aptamer. In acrystallography investigation of the human IgG-aptamer complex it wasfound that this optimized aptamer interacts with several conservedresidues of the human IgG Fc domain mainly via van der Waals contactsand hydrogen bonds rather than electrostatic forces. Moreover, it wassuggested that the binding structure of the aptamer can be stabilized bycalcium ions and that binding can be reversed through calcium chelation.However, despite these valuable fundamental studies of an anti-human Fcaptamer, there remains a need for robust methods that facilitate humanFc capture so that more detailed and more accurate analyses can beperformed during pharmacokinetic, pharmacodynamic and biotransformationstudies.

With this technology, a novel, aptamer-based sample preparation methodis provided for the selective capture of human immunoglobulin Fcdomains. A 23-nucleotide aptamer with the any one of SEQ ID NO 1-13 canbe employed. In addition, other sequences described in U.S. Pat. No.8,637,656 can be used and are incorporated herein by reference in theirentirety. Namely, SEQ ID NOs 2-13 have been shown to have a capacity forthe selective capture of human immunoglobulin and are equivalent intheir applicability to SEQ ID NO 1 with respect to the instanttechnology. Some of the sequences described in U.S. Pat. No. 8,637,656may lack the species selectivity necessary to achieve selective captureof human Fc domains from animal, pre-clinical biofluids. These aptamersequences might mimic the comparatively broad specificity of protein A.Nevertheless, it is believed they too will be amenable to the methods ofthe instant invention, potentially for an alternative means for thepurification of monoclonal antibodies from cell culture media. Each ofSEQ ID Nos 1-13 are shown below.

SEQ ID NO 1:5′ terminus - G G rA rG rG [i2FU] rG C [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

SEQ ID NO 2:5′ terminus - G G rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] [i2FC] G A A A rG rG rA rA [i2FC] [i2FU]C C - 3′ terminus

SEQ ID NO 3:5′ terminus - G G rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] C G A A A rG rG rA rA [i2FC] [i2FU] C C- 3′ terminus

SEQ ID NO 4:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus

SEQ ID NO 5:5′ terminus - G G rA rG rG [i2FU] rG C U(OMe) C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

SEQ ID NO 6:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] U(OMe) [i2FC] [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus

SEQ ID NO 7:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] C(OMe) [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus

SEQ ID NO 8:5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] C(OMe) G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe)- 3′ terminus

SEQ ID NO 9:5′ terminus - G G rA rG [i2FG] [i2FU] rG C [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

SEQ ID NO 10:5′ terminus - G G rA rG rG [i2FU] [i2FG] C [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

SEQ ID NO 11:5′ terminus - G G rA rG rG [i2FU] rG rC [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

SEQ ID NO 12:5′ terminus - G G rA rG rG [i2FU] rG C rU C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′ terminus

SEQ ID NO 13:5′ terminus - G G rA rG rG [i2FU] rG C U(OMe) C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus

where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.U(OMe), C(OMe), G(OMe) and A(OMe) represent 2′-methoxy substitutednucleotides.

In all embodiments of this technology, the aptamer ligand is immobilizedto substrates suitable for performing affinity capture and sampleenrichment. Substrates include, but are not limited to, the surfaces ofmicrovolume plates, pipet tips, and labware as well as both porous andnon-porous resins comprised of polymers, silica and organosilica.

When an aptamer is immobilized onto a resin, the resin can then beimmobilized onto a consumable (e.g, microvolume plates, pipet tips,and/or other labware). In this way, the resin can be used to increasethe surface area and/or amount of aptamer bound to the surface. In otherembodiments, the resin having an immobilized aptamer can be used as astationary phase in a chromatography column.

The sequence of aptamer can be modified from either its 5′- or 3′-terminus or one of its internal residues. This sequence modification canimpart a chemical handle and an optional spacer arm moiety, includingbut not limited to hydrophilic molecular compositions, such as 2 to 50repeat units of polyethylene glycol or polyethylene oxide. In addition,this sequence modification can be used for the sake of achievingoriented immobilization. For instance, with a primary amino group addedto either the 5 or 3′ terminus, the aptamer can be modified via anucleophilic/electrophilic reaction scheme to yield a single-pointlinkage to another molecule, surface, or resin that would also impartdefined directionality. In some embodiments, the above describedchemical handle can include a biotin, amine, carboxyl or thiol group.Upon immobilization and storage, the immobilized aptamer can beaugmented with one or more nuclease inhibitors to improve its shelflife. Nucleases can also be added in storage solutions and workingbuffers to extend the stability of immobilized aptamer.

FIG. 1 shows a method 100 of capturing human immunoglobulin Fc domainsin a biofluid sample. The method includes providing an affinity capturedevice 105. The affinity capture device has a surface having an aptamerthat is at least 80% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ IDNO 12, or SEQ ID NO 13) immobilized onto the surface of the affinitycapture device. The aptamer can be 85%, 90%, 95%, 98%, or 99% identicalto SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5,SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ IDNO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). Theaptamer can be a 23 nucleotide aptamer. The aptamer can be a 23nucleotide aptamer having an extended or truncated sequence of about 3or about 5 residues. The aptamer can be non-covalently or covalentlyimmobilized onto the surface of the affinity capture device.

The method 100 also includes diluting the biofluid sample with a bindingbuffer 110. The binding buffer includes (A)tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES),2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The method 100 also includes adsorbing the humanimmunoglobulin Fc domains 115 in the biofluid sample to the aptamer withthe binder buffer.

The binding characteristics of the aptamer are highly dependent on thecomposition of the buffer systems with which it is employed (See FIGS.2A-5 ). Based on investigative work, it has been found that the aptamerfunctions with a metal-dependent mechanism. In using the aptamer, it hasbeen observed that the presence of an appropriately selected divalention is essential for maintaining high affinity and selectivity (FIGS. 2Aand 2B, FIG. 5 ). Nakamura and co-workers made the proposal that calciumions are the most critical component to forming the affinity competentmotif of this aptamer. Herein, it is shown that magnesium ions are moreeffective in increasing the binding capacity of the aptamer withoutintroducing any significant amount of non-specific binding with otheruntargeted proteins. The concentration of magnesium cation can bebetween 50 µM to about 1 mM.

As used herein, the term “about” means that the numerical value isapproximate and small variations would not significantly affect thepractice of the disclosed embodiments. Where a numerical limitation isused, unless indicated otherwise by context, “about” means the numericalvalue can vary by ±10% and remain within the scope of the disclosedembodiments

Meanwhile, monovalent ions have been found to serve as a disruptor tothe affinity binding motif, serving as potential alternative eluents tothe chelator proposed by Nakamura and co-workers. Accordingly, in someembodiments of this technology, a monovalent cation solution, includingbut not limited to solutions of ammonium, sodium and potassium, can beused in the form of an elution buffer to aid the recovery of targetprotein/analyte at neutral pH (FIGS. 3A, 3B, 4A, 4B, and 6 ). Moreover,in contrast to the phosphate buffers proposed by Nakamura andco-workers, it has been found that the aptamers of this invention workmore advantageously in Tris buffer. In fact, a Tris buffer was found toyield better binding capacity versus a phosphate buffer when used withthe same ion additives. Triethylamine (TEA), MES (2-ethanesulfonicacid), and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)can confer comparable advantages over a phosphate buffer.

In some embodiments, the binding buffer has a concentration ofmonovalent cations less than about 50 mM. The binding buffer can have aconcentration of monovalent cations less than about 30 mM. In someembodiments, the pH of the binding buffer is between about 5 and about9. The pH of the binding buffer can be between about 6 to about 8. ThepH of the binding buffer can be about 7.2. For example, the pH of thebinding buffer can be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, or 9.0. These pH values can be used to form a range, forexample, from about 7.0 to about 7.4 or from about 7.1, to about 7.3.

The biofluid sample can be diluted. For example, the biofluid sample canbe diluted by a factor of 2, 10, or 20. In some embodiments, thebiofluid sample is diluted by a factor of 1, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The dilution factors can beused to form a range, for example, from about 2 to about 20 or fromabout 2 to about 10, or from about 10 to about 20. In some embodiments,the biofluid sample is diluted to obtain a total monovalent cationconcentation of the biofluid sample of no greater than 100 mM, nogreater than 50 mM or no greater than 30 mM. The total monovalent cationconcentration of the diluted biofluid sample can be no greater than 100mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, or 10 mM.

Referring to FIG. 1 , the method 100 can also include eluting theadsorbed himan immunoglobulin Fc domain from the immobilized aptamerusing an eluent 120. The eluent has an ammonium concentration betweenabout 10 mM to about 1000 mM. The ammonium is in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The ammonium concentration can be, for example, between about50 mM to about 500 mM. The pH of the eluent can be between about 6.5 toabout 8.0.

The method 100 can also include washing the adsorbed humanimmunoglobulin Fc domains with the binding buffer 125. In someembodiments, the washing is done with a buffer comprising Ca⁺.

The method 100 can also include analyzing the eluted humanimmunoglobulin Fc domain 130 with a detector. The detector can be asandwiched enzyme linked immunosorbent assay or a mass spectrometer.

In some embodiments, the human immunoglobulin Fc domains are containedwithin a humanized monoclonal antibody and the method also includespurifying the humanized monoclonal antibody (not shown).

The technology also relates to a method of eluting a humanimmunoglobulin Fc domain from an immobilized aptamer. The methodincludes providing an affinity capture device having a surface having anaptamer that is at least 80% identical to SEQ ID NO 1 immobilized ontothe surface of the affinity capture device. The immobilized aptamer hasan adsorbed human immunoglobulin Fc domain. The adsorbed humanimmunoglobulin Fc domain is eluted from the immobilized aptamer using aneluent having an ammonium concentration between about 10 mM to about1000 mM. The ammonium is in the form of tetramethylammonium,triethylammonium, ammonium formate, or ammonium acetate. The method caninclude any of the embodiments described herein.

The technology also relates to kits. The kits can include an affinitycapture device comprising a surface having an aptamer that is at least80% identical to SEQ ID NO 1 immobilized onto the surface of theaffinity capture device. The kit can also include a vial of a bindingbuffer including (A) tris(hydroxymethyl)aminomethane (Tris),trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM. The kit can include any of the embodiments describedherein. This kit can also include a vial of eluent having an ammoniumconcentration between about 10 mM to about 1000 mM. The ammonium can bein the form of tetramethylammonium, triethylammonium, ammonium formate,or ammonium acetate. The kit can also include a vial of buffercomprising Ca⁺. The buffer provided in the kit can be concentrated andthe kit can include instructions for diluting the concentrated buffer toa working concentration. The buffer can be diluted using water. The kitcan also contain instructions for performing any one of the methodsdescribed herein.

The technology relates to a kit that includes an affinity capture devicecomprising a surface having an aptamer that is at least 80% identical toSEQ ID NO 1 immobilized onto the surface of the affinity capture deviceand a vial of an eluent. The eluent has an ammonium concentrationbetween about 10 mM to about 1000 mM. The ammonium can be in the form oftetramethylammonium, triethylammonium, ammonium formate, or ammoniumacetate. The kit can include any of the embodiments described herein.The kit can include instructions for performing any of the methodsdescribed herein.

As reduced to practice, an anti-human Fc aptamer can be used withcarefully defined protocol steps, namely binding, wash and elutionsteps. Specifically, for the binding step, the aptamer can be used witha Tris, TEA, MES or HEPES binding buffer comprised of Mg at aconcentration ranging from 10 µM to 20 mM or more ideally from 50 µM to1 mM. The concentration of monovalent cation is purposely kept minimalin this binding buffer to a concentration of less than 50 mM, moreideally less than 30 mM. In addition, the pH of the solution is designedto be between 5 and 9, more ideally 6 to 8. With this formulation andmethod, the binding capacity of the aptamer is improved without loss ofselectivity (See FIGS. 5 and 6 ). This stands in contrast to the examplepresented by Nakamura and co-worker, wherein a buffer of 145 mM NaCl,5.4 mM KCl, 1.8 mM CaCl₂. 0.8 mM MgCl₂, 20 mM Tris, pH 7.6 wasdescribed. Because it is critical to minimize the presence of anymonovalent ions during the binding step of the aptamer capture protocol,certain embodiments of this invention entail a 2 to 20-fold dilution ofsample. In this way, a complex matrix (e.g. biofluids) is reduced in itsmonovalent cation concentration and the negative impact of endogenousmonovalent ions is minimized. (FIGS. 8A-9C).

In exemplary embodiments, the method includes a wash step comprised oftwo procedures. The first wash procedure is performed exclusively withwashing the adsorbed analyte and resin with the binding buffer. Thesecond wash procedure preferentially performed with a Ca²⁺ containingbuffer, since it has been observed to significantly decrease thenon-specific binding of the aptamer ligand in complex sample matrix andto improve the purity of the captured/enriched hIgG (FIGS. 9A-9C).

In the final step of this method, the captured Fc domain can optionallybe eluted by using neutral pH buffers (pH between 6.5-8.0) withchelating agents, including those described by Nakamura and co-workers,like 10 to 200 mM EDTA, malate, citrate and porphine, or withconcentrated monovalent cation like K⁺, Na⁺, Ag⁺ and Cs⁺. Alternatively,the Fc domain containing analyte can be eluted with a novel buffersystem, as disclosed herein, that are based on a bisphosphonate,including but not limited to medronic and etidronic, or with anMS-compatible, volatile monovalent cation, such as ammonium,tetramethylammonium or triethylammonium. In one embodiment, an elutionbuffer composed of 10 mM to 1000 mM, or more ideally 50 to 500 mM,ammonium formate or ammonium acetate is employed. With this type ofeluent, it is possible to perform online immunoaffinity MS assays. Aswell, common protein A elution buffers with acidic pH (pH ≤ 4.0), like0.1 M glycine HCl, 0.1 M citric acid, 5% acetic acid or formic acid,make for another option for eluting the capture Fc domain analyte. Theseselections of elution condition extend the compatibility of aptameraffinity capture with many downstream processes. In some embodiments,the anti-human Fc aptamer and corresponding devices are employed toperform sample preparation for a subsequent LC or LCMS based assay, likereversed phase chromatography, ion exchange, hydrophilic interactionliquid chromatography, hydrophobic interaction chromatography, proteinAn affinity chromatography, subunit analysis, peptide mapping, glycanprofiling and intact mass analysis. Analyses facilitated by thistechnology can also include online immunoaffinity capture, capillaryelectrophoresis, and mass spectrometry. Additionally, the methodsdescribed herein can be applied to enzyme linked immunosorbent assays(Example 7). In another embodiment, a heat-induced denaturation of theaptamer can be applied to elute captured human Fc analyte withoutintroducing any elution buffer.

In summary, this technology prescribes a novel sample preparation methodfor the selective capture of human Fc domains. Being that it is based onthe use of a small affinity ligand, it shows advantageous bindingcapacity versus methods dependent on a full-size IgG antibody ligand andclear improvements over the prior art established by Nakamura andco-workers. (FIG. 7 ).

EXAMPLE 1: Capture of hIgG Using Different Binding Buffers

An aptamer, having SEQ ID NO 1, was adapted from US 8,637,656 B2 andmade to contain a biotin tag at its 5′-terminus. Synthesis of theaptamer was performed by Integrated DNA Technologies (Coralville, IA).Each 5 µg of biotinylated aptamer was immobilized to 40 µL of highcapacity streptavidin resin (commercially available from Thermo FisherScientific, San Jose, CA) in a 96-well filter plate (commerciallyavailable from MilliporeSigma, Burlington, MA) via a 30 minuteincubation at room temperature. A positive pressure manifold(commercially available from Waters Technologies Corporation, Milford,MA) was utilized to drive flow through the filter plate. The amount ofaptamer immobilized was determined by abosorbance at 260 nm using aDS-11 spectrophotometer (commercially available from DeNovix Inc.,Wilmington, DE). This non-covalently immobilized aptamer affinity resinwas used to perform the affinity capture of 100 µg human IgG (IgG fromhuman serum, commercially available from Sigma-Aldrich (nowMilliporeSigma), St. Louis, MO) contained within 200 µL of severaldifferent binding buffers (specified below). After 1 hour of mixing, theresin was washed with two repeated 200 µL volumes to remove any unboundprotein. Combined hIgG flow-through and washing fractions were saved todetermine the amount of hIgG breakthrough. Captured human IgG was elutedwith two repeat 200 µL volumes of elution buffer (200 mM EDTA, 10 mMtris, pH 7.2). The eluate was saved for hIgG recovery measurements usinga fluorescence plate reader (Ex. 280 nm, Em. 370 nm; Gemini™ XPS platereader, commercially available from Molecular Devices, San Jose, CA).

The binding capacity of the aptamers, incorporated herein by reference,are highly dependent on the composition of binding buffer. Toinvestigate the impact of divalent ions on aptamer bindingcharacteristics, a series of Tris buffers containing different divalentcations were screened. The binding buffers tested were: 10 mM Trisbuffer with 5 mM MgCl₂, pH 7.2; 10 mM Tris buffer with 5 mM CaCl₂, pH7.2; 10 mM Tris buffer with 5 mM MnCl₂, pH 7.2, and 10 mM Tris bufferwith 5 mM Zn acetate, pH 7.2. Tris buffer with no added cations (10 mMTris/Tris HCl, pH 7.2) was used as a control and compared against thebuffer system proposed by Nakamura and co-workers (145 mM NaCl, 5.4 mMKCl, 1.8 mM CaCl₂. 0.8 mM MgCl₂, 20 mM Tris, pH 7.6). As can be seen inFIGS. 2A and 2B, the presence of divalent cations, especially Zn²⁺ andMg²⁺, leads to significantly improved capture and recovery of hIgG.Those two buffers stand out as exemplary embodiments for both theirrecovery and selectivity. On the other hand, it was surprising to noticethat only 20% of loaded hIgG was captured when the buffer proposed byNakamura and co-workers was employed (comparative example), which meansthe presence of concentrated sodium chloride (NaCl) and the combinationof multiple divalent/monovalent cations in buffer inhibit the bindingcapacity of aptamer.

That there is such a negative impact on hIgG capture from the presenceof monovalent cations was again demonstrated by comparing bindingcapacity and protein recovery using binding buffers both containing andnot containing Na⁺ or K⁺ cations. The binding buffers explored in thisparticular study included a) buffers without 150 mM NaCl: 10 mM Trisbuffer with 5 mM MgCl₂, pH 7.2; 10 mM Tris buffer with 5 mM CaCl₂, pH7.2; 10 mM Tris buffer with 5 mM MnCl₂, pH 7.2, and 10 mM Tris bufferwith 5 mM Zn acetate, pH 7.2 versus b) matching buffers made to alsocontain 150 mM NaCl as well as 10 mM Tris buffer with 5 mM KCl, 150 mMNaCl, pH 7.2. Tris buffer containing 150 mM NaCl was also used as acontrol. The results of this study are summarized in FIGS. 3A and 3B.From these data, it appears that the presence of 150 mM NaCl in adivalent cation containing binding buffer is disruptive to theadsorption of hIgG onto the immobilized aptamer, as is manifest in asignificant decrease in aptamer binding capacity and hIgG recovery.Adding potassium cations (K) into the sodium chloride (NaCl) containingbinding buffer had no effect toward improving recovery. This result isconsistent with the observed binding characteristics of the aptamer whenusing the binding buffer proposed by Nakamura and co-workers, being thatit contains relatively high concentrations of monovalent cations- thatis, monovalent cations at concentrations greater than 10 mM.

The type of aptamer binding buffer is yet another factor that affectscapture efficiency. Phosphate-based buffers were used here asalternatives for the binding step. Three phosphate buffers were appliedin this study including: 20 mM Na₂HPO₃ with no added NaCl, pH 7.0; 20 mMNa₂HPO₃ with 150 mM NaCl, pH 7.0; and 20 mM Na₂HPO₃ with 5 mM MgCl₂, pH7.0. 10 mM Tris buffer with the same ionic components were employed as acontrol. As shown in FIGS. 4A and 4B, Tris buffer provided betterbinding capacity than phosphate buffer when used with the same ionadditives. When the aptamer was used with phosphate-only buffer, itshowed almost no affinity for IgG, while its affinity could be partiallyrecovered via the addition of 5 mM Mg²⁺. According to the reportedcrystal structure of the hIgG-aptamer complex pertinent to thistechnology, it is reasonable to suggest that phosphate might interruptthe folding of the aptamer into its active affinity motif.

In summary, 10 mM Tris buffer containing divalent cations like Zn²⁺ orMg²⁺ stand out as exemplary embodiments of this technology. Additionalexperiments have been performed to demonstrate the usability of thesebuffers and their results are detailed below.

Example 2: Impact of Binding and Elution Buffer on Aptamer SelectiveCapture

The presence of divalent cations in the binding buffer has the potentialto facilitate the activation of the aptamer affinity motif, yet it couldalso instigate non-specific binding. Accordingly, we also aimed toaddress the non-specific binding of the aptamer. The biotinylatedaptamer was immobilized as described in Example 1 and subsequently usedfor hIgG capture. Different divalent cation containing Tris bindingbuffers (Mg, Ca, Mn, Zn) were prepared with and without 150 mM NaCl andthereafter employed in this study. The compositions of these binding andelution buffers were listed earlier in Example 1. Rabbit IgG (rIgG)(commercial available from Sigma-Aldrich (now MilliporeSigma), St.Louis, MO) and bovine serum albumin (BSA) (commercial available fromSigma-Aldrich (now MilliporeSigma), St. Louis, MO) were used as twonegative controls to investigate the non-specific binding of theaptamer. 100 µg of hIgG, rIgG and BSA were loaded to the non-covalentlyimmobilized aptamer resin using 200 µL volumes of the various bindingbuffers. After binding and washing steps, captured proteins were elutedwith elution buffer and protein recovery in each eluate was measuredusing a fluorescence plate reader.

The results on protein recovery are summarized in FIG. 5 .Interestingly, buffer containing Zn²⁺ brought about a high level ofnon-specific binding with all proteins tested, especially when no NaClwas present. Therefore, Zn containing binding buffer was deemed to beunsuitable for this method. The same issue was observed with Mn²⁺containing buffer, which also produced a high level of non-specificbinding with rIgG. In light of these results, it should be noted thatsome NaCl can be included in a wash buffer so as to attenuate some typesof non-specific binding.

Further analysis of the results shows that a buffer comprised of Mg²⁺ isthe most promising for binding, as it provided effective capture withnegligible non-specific binding. Nevertheless, it can be shown thathaving different concentrations of Mg²⁺ in the binding buffer can be ofimpact to binding capacity. As summarized in FIG. 6 , a binding bufferwith Mg²⁺ concentration between 50 µM to 1 mM resulted in the highesthIgG capture and recovery among all conditions tested. It seems,therefore, that the use of a binding buffer with more than 20 mM or lessthan 10 µM Mg²⁺ might result in sub-optimal hIgG binding.

In another investigation, we chose to investigate the use ofconcentrated ammonium acetate buffer for hIgG elution (FIG. 6 ). Sinceour preliminary results indicated that binding is highly dependent onthe presence of certain divalent cations, we aimed to see if a competingcation can be used to displace the divalent cation from the hIgG-aptamercomplex and thereby elute the captured hIgG. As proposed by Nakamura, anEDTA buffer can be used as an elution buffer. However, this non-volatilechelating agent can cause problems for downstream mass spectrometric(MS) analysis. In contrast, a buffer with a competing volatilemonovalent cation like ammonium acetate (NH₄OAc), ammonium formate orammonium bicarbonate can be a more MS friendly elution buffer. To thispoint, 200 mM ammonium acetate, pH 7.2 was used to elute captured hIgGfrom the aptamer and the quality of the eluate was compared to thatobtained with 200 mM EDTA elution buffer. As shown in FIG. 6 , theelution efficiency of concentrated NH₄OAc was highly dependent on Mg²⁺concentration in the binding buffer. For binding buffers comprised of 50µM to 10 mM Mg²⁺, the recovery of hIgG with NH₄OAc can be 80-90% of thatobtained using an EDTA elution buffer.

In summary, 10 mM Tris binding buffer with Mg²⁺ can minimize undesirablenon-specific binding and provide exemplary binding capacity. The idealMg²⁺ concentration in the binding buffer is between 10 µM and 10 mM,more ideally 50 µM to 1 mM. Importantly, with this selected bindingbuffer, concentrated ammonium buffer (e.g. 200 mM ammonium acetate) canbe used to achieve comparatively high recovery with an MS-friendlyelution buffer.

Example 3: Evaluation of Aptamer Binding Capacity

To determine the binding capacity of the aptamer, protocols establishedwith this technology were employed to sample preparations involvingamounts of hIgG exceeding 500 µg. 5 µg, 12.5 µg, 25 µg, 37.5 µg, 50 µgand 100 µg of biotinylated aptamer were immobilized onto 40 µL ofstreptavidin resin. Procedures for aptamer immobilization and hIgGcapture were identical to those described in previous examples. Therecovery of hIgG was plotted against the amount of aptamer immobilizedto estimate the maximum binding capacity of the aptamer (FIG. 7 ). 10 mMTris, 0.5 mM Mg, pH 7.2 and 200 mM EDTA, 10 mM Tris, pH 7.2 wereemployed for binding and elution, respectively. According to the hIgGrecovery curve, it can be seen that by using the optimized buffers andprotocols of this technology, the maximum binding capacity of aptamer isclose to 6.5 µg hIgG/µg immobilized aptamer, which is about 20 timeshigher than that estimated to be possible with a full-length IgG-basedanti-human Fc antibody. It is also worth mentioning that the surfacecoverage of the aptamer was constrained in this testing by thelimitations of noncovalent immobilization to a streptavidin resin. Ahigher binding capacity can be predicted for an aptamer prepared by wayof direct immobilization. The coverage of the immobilized aptamer can beabout 1 to about 1000 nmol/m². To minimize costs, lower coverageimmobilization might be preferred. Yet, to achieve high binding capacitysurfaces, higher coverages are desirable.

In yet another example, the capabilities of this aptamer-based affinitycapture has been compared to a commercialized nanobody ligand for humanFc capture. A nanobody derived from a camelid V_(H)H domain waspreviously developed and is now commercially available in the form ofThermo-Fisher Scientific CaptureSelect® branded technology. In thisstudy, anti-human Fc V_(H)H ligand was recombinantly expressed(GenScript, Piscataway, NJ) from a sequence adapted from US 9,040,666 B2(incorporated by reference herein in its entirety) and designed to havea C-terminal extension consisting of an extra cysteine residue, TEVprotease cleavage site and His-tag. The obtained nanobody was treatedwith ProTEV (commercially available from Promega, Madison, WI) to removeits His-Tag and thereafter derivatized with EZ-Link™, idoacetyl-PEG2Biotin (commercially available from Thermo-Fisher Scientific, San Jose,CA). The purity of the resulting biotinylated V_(H)H was assessed byLCMS and determined to be greater than 80% biotinylated. BiotinylatedV_(H)H was non-covalently immobilized to 40 µL of streptavidin resinslurry and used to capture hIgG using the same procedure as the aptamer.The amount of V_(H)H immobilized was measured by absorbance at 280 nmusing a DS-11 spectrophotometer (commercially available from DeNovixInc., Wilmington, DE). 20 mM phosphate, pH 7.0 and 0.1 M glycine-HCl, pH2.7 was recommended by CaptureSelect literature from Thermo FisherScientific as being appropriate binding and elution buffers. As shown inFIG. 7 , the maximum binding capacity of this V_(H)H based ligand (5 µghIgG/ µg immobilized V_(H)H) was lower than that of the aptamer.

Example 4: Capture of hIgG From Biofluid

To investigate the selectivity of purifying human Fc domains frombiofluid matrices, different amounts of NIST mAb (NIST ReferenceMaterial 8671) were spiked into rat plasma, followed by affinity capturewith non-covalently immobilized aptamer using the protocols described bythis invention. The purity of flow-through, washing and eluate fractionswas analyzed using an ACQUITY® UPLC® H-Class Bio (commercially availablefrom Waters Technologies Corporation, Milford, MA) with an ACQUITY®BEH300 C4 column (1.7 µm, 2.1×100 nm) (commercially available fromWaters Technologies Corporation, Milford, MA). The protein concentrationof each fraction was determined using a fluorescence plate reader(Gemini XPS, (commercially available from Molecular Devices, LLC, SanJose, CA); Ex. 280 nm, Em. 370 nm) and re-calculated according to thechromatographic purity assessment. FIGS. 8A-C shows example data fromthe purification of 100 µg of NIST mAb from 20 µL rat plasma matrixusing 12.5 µg of immobilized aptamer. After purification, about 85% ofthe spiked NIST mAb was recovered with purity increasing from 10.6% to72.4%.

The small portion of impurity in the purified NIST mAb eluate wasdetermined to be the non-specific binding of rat plasma endogenousproteins to the aptamer. The competition in binding between NIST mAb andplasma protein was investigated by loading 20 µL of rat plasmacontaining 50 µg of NIST mAb onto an increasing amount of immobilizedaptamer (5 µg, 12.5 µg, 25 µg). The result of this experiment issummarized in FIGS. 8D-E. From which, it is possible to observe that thebinding ratio of NIST mAb to plasma protein is close to 7:3 (from UVmeasurement) for the current experimental approach. These data suggestthat non-specific binding can be further reduced.

Hence, an extra washing step (second washing) was applied before theelution step. To test out this optional protocol step, 20 µL of plasmaand 25 µg of NIST mAb were diluted with binding buffer to 200 µL thenloaded onto 40 µL of streptavidin resin wherein 5 µg of aptamer wasnon-covalently immobilized. After the first washing with binding buffer,two 200 µL volumes of each potential second washbuffers was applied,including the binding binding buffer proposed by Nakamura and co-workers(10 mM Tris, 5 mM CaCl₂, pH 7.2); 20 mM phosphate, pH 7.0; 0.1 Mglycine-HCl, pH 2.7; 150 mM NaCl and binding buffer (control). Elutionwas thereafter performed with an EDTA buffer. Herein, the second washingand elution fractions were collected and analyzed by LC-UV analysis. Asshown in FIG. 9A, neither the binding buffer proposed by Nakamura et alnor 20 mM phosphate, 0.1 M glycine and 150 mM NaCl were found to besuitable secondary washing buffers. In both cases, the captured NIST mAbwas eluted together with plasma impurities. On the other hand, a Trisbuffer containing 5 mM CaCl₂ was able to selectively wash off impuritiesfrom the aptamer. According to FIGS. 9B and 9C, the immobilized aptamershowed the best NIST mAb recovery, together with the highest purity,after the application of a secondary washing step based on 5 mM CaCl₂containing tris buffer. Therefore, this Ca²⁺ containing buffer is arecommended, yet optional, secondary washing buffer for use in anoptimized protocol for capturing human Fc domains from biofluid samples.An exemplary embodiment of this invention is outlined in followingprotocol.

Example 5: Exemplary Protocol for the Capture of a Humanized mAb BasedTherapeutic Protein From a Preclinical Type Biofluid Sample

This exemplary protocol is intended for use with an immobilized aptamermaterial or device.

-   Buffers used:    -   Binding/ 1^(st) washing buffer: 10 mM Tris, 0.5 mM MgCl₂, pH 7.2    -   2^(nd) (secondary) washing buffer: 10 mM Tris, 5 mM CaCl₂, pH        7.2    -   Elution buffer: 10 mM Tris, 200 mM EDTA, pH 7.2, or 200 mM        Ammonium acetate, pH 7.2 (for MS analysis)-   Biofluid sample preparation:    -   It is recommended to dilute a biofluid sample at least ten-fold        with binding buffer before loading sample onto the immobilized        aptamer. For example, dilute 10 µL of biofluid sample with 90 µL        binding buffer to make a final volume of 100 µL.-   Binding step:    -   Equilibrate the immobilized aptamer resin/device with binding        buffer (~10 column volumes (CV) versus the volume of affinity        resin/bed)    -   Load diluted biofluid sample onto the immobilized aptamer    -   Incubate device at room temperature for 1 hour with mixing.        (e.g. plate shaker at 5-600 rpm)    -   Remove flow-through solution from resin/device-   Washing step:    -   Perform the 1^(st) washing step using binding buffer (~10CV of        resin used)    -   Remove 1^(st) washing solution from the resin/device    -   Perform the 2^(nd) washing step using the secondary washing        buffer (~10 CV of resin used).    -   Remove the secondary wash solution from the resin/device-   Elution step:    -   Elute captured human Fc analyte with desired elution buffer (~10        CV of resin can be used, though the volume of elution buffer can        be varied as needed)    -   Save fractions as eluate for downstream analysis

Example 6: Online Immunoaffinity Capture MS Analysis Example

Immobilized anti-human Fc aptamer can be employed for onlineimmunoaffinity capture MS to facilitate the identification andquantitation of humanized biotherapeutics. In some embodiments, animmunoaffinity column is packed with immobilized aptamer resin andcoupled directly with LCMS for analysis. A solvent manager can be usedin this application to deliver binding buffer, the secondary washingbuffer and MS compatible elution buffer. Elution buffer described hereincludes but is not limited to volatile monovalent salts like ammoniumformate, ammonium acetate, ammonium bicarbonate, tetramethylammonium ortriethylammonium; volatile acids like formic acid, acetic acid, TFA orDFA; volatile solvents like acetonitrile and methanol. To avoidcontamination of MS, LC flow-through can be diverted to waste forbinding and washing steps and then switched back to MS for the elutionstep. Depending on LC settings, the first 30 seconds to 2 minutes ofeffluent from the immunoaffinity column can also be directed to waste.Alternatively, an MS-compatible washing buffer can be employed. Uponelution and detection by MS, retention time, peak area and m/z valuesare used together to characterize components of the sample. In anotherembodiment, the packed aptamer immunoaffinity column can be applied to atwo-dimensional (2D) LCMS system for more comprehensive analyses. Asecondary analytical column involved in such a 2D-LCMS system can beequipped with a multitude of techniques, including but not limited to,normal phase separation, reverse phase separation, size-exclusiveseparation, ion-exchange separation, HILIC separation, HIC separation,affinity capture or enzymatic digestion.

Example 7: Anti-Human Fc ELISA Example

Being an anti-human Fc affinity ligand, the aptamer sequencesincorporated herein by reference can be used along with aspects of thisinvention to establish a sensitive enzyme linked immunosorbent assay(ELISA) for accurate quantification of human IgG or humanizedtherapeutics with Fc domain. In some embodiments, anti-human Fc aptameris coated onto the surface of an ELISA plate and used to capture targetIgG from biofluids. The selected binding buffer described above isapplied to ensure that a desired binding capacity and specificity areachieved. Unbound impurities are then washed from the plate using one ormore of the described washing buffers. In a typical embodiment, asecondary detection ligand with enzyme label, such as horse radishperoxidase, is utilized to create a sandwich immunoassay. This detectionligand binds to human IgG at one or more domain, including but notlimited to CH1, light chain κ, light chain λ, VH or VL. After binding ofthe detection ligand, a substrate, usually TMB, is added to the ELISAplate and catalyzed by the enzyme label to produce a colored productthat the concentration can be measured using a UV plate reader. In analternative embodiment, the aptamer ligand is free in solution yetmodified to contain an enzyme label. A direct ELISA measurement can thenbe performed with the binding protocols described by the instanttechnology. In yet another embodiment, the aptamer ligand is modified tocontain the enzyme label itself so as to facilitate an indirect ELISAmeasurement.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents were consideredto be within the scope of this technology and are covered by thefollowing claims. The contents or all references, issued patents, andpublished patent applications cited throughout this application arehereby incorporated by reference.

SEQUENCE LISTING TABLE SEQ ID NO: DESCRIPTION SEQUENCE 1 23-nucleotideaptamer 5′ terminus -G rA rG rG [i2FU] rG C [i2FU] C C G A A A rG rG A A[i2FC] [i2FU] C C - 3′ terminus where A, C, G, U represent fournucleobases. Uppercase letter corresponds to deoxyribonucleotide, and rXindicates ribonucleotide. [i2FC] and [i2FU] denote 2′-fluoro substitutedpyrimidine nucleotides 2 23-nucleotide aptamer 5′ terminus - G G rA rGrG [i2FU] rG [i2FC] [i2FU] [i2FC] [i2FC] G A A A rG rG rA rA [i2FC][i2FU] C C - 3′ terminus where A, C, G, U represent four nucleobases.Uppercase letter corresponds to deoxyribonucleotide, and rX indicatesribonucleotide. [i2FC] and [i2FU] denote 2′-fluoro substitutedpyrimidine nucleotides. 3 23-nucleotide aptamer 5′ terminus - G G rA rGrG [i2FU] rG [i2FC] [i2FU] [i2FC] C G A A A rG rG rA rA [i2FC] [i2FU] CC - 3′ terminus where A, C, G, U represent four nucleobases. Uppercaseletter corresponds to deoxyribonucleotide, and rX indicatesribonucleotide. [i2FC] and [i2FU] denote 2′-fluoro substitutedpyrimidine nucleotides. 4 23-nucleotide aptamer 5′ terminus - G(OMe)G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] [i2FC] G(OMe) A(OMe)A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) 3′ terminus whereA, C, G, U represent four nucleobases. Uppercase letter corresponds todeoxyribonucleotide, and rX indicates ribonucleotide. [i2FC] and [i2FU]denote 2′-fluoro substituted pyrimidine nucleotides. C(OMe), G(OMe) andA(OMe) represent 2′-methoxy substituted nucleotides. 5 23-nucleotideaptamer 5′ terminus - G G rA rG rG [i2FU] rG C U(OMe) C C G A A A rG rGA A [i2FC] [i2FU] C C - 3′ terminus where A, C, G, U represent fournucleobases. Uppercase letter corresponds to deoxyribonucleotide, and rXindicates ribonucleotide. [i2FC] and [i2FU] denote 2′-fluoro substitutedpyrimidine nucleotides. U(OMe) represents 2′-methoxy substitutednucleotides. 6 23-nucleotide aptamer 5′ terminus - G(OMe) G(OMe) rA rGrG [i2FU] rG [i2FC] U(OMe) [i2FC] [i2FC] G(OMe) A(OMe) A(OMe) A(OMe) rGrG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus where A, C, G, Urepresent four nucleobases. Uppercase letter corresponds todeoxyribonucleotide, and rX indicates ribonucleotide. [i2FC] and [i2FU]denote 2′-fluoro substituted pyrimidine nucleotides. U(OMe), C(OMe),G(OMe) and A(OMe) represent 2′-methoxy substituted nucleotides. 723-nucleotide aptamer 5′ terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG[i2FC] [i2FU] C(OMe) [i2FC] G(OMe) A(OMe) A(OMe) A(OMe) rG rG rA rA[i2FC] [i2FU] C(OMe) C(OMe) - 3′ terminus where A, C, G, U representfour nucleobases. Uppercase letter corresponds to deoxyribonucleotide,and rX indicates ribonucleotide. [i2FC] and [i2FU] denote 2′-fluorosubstituted pyrimidine nucleotides. C(OMe), G(OMe) and A(OMe) represent2′-methoxy substituted nucleotides. 8 23-nucleotide aptamer 5′terminus - G(OMe) G(OMe) rA rG rG [i2FU] rG [i2FC] [i2FU] [i2FC] C(OMe)G(OMe) A(OMe) A(OMe) A(OMe) rG rG rA rA [i2FC] [i2FU] C(OMe) C(OMe) - 3′terminus where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.C(OMe), G(OMe) and A(OMe) represent 2′-methoxy substituted nucleotides.9 23-nucleotide aptamer 5′ terminus - G G rA rG [i2FG] [i2FU] rG C[i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′ terminus where A, C,G, U represent four nucleobases. Uppercase letter corresponds todeoxyribonucleotide, and rX indicates ribonucleotide. [i2FC] and [i2FU]denote 2′-fluoro substituted pyrimidine nucleotides. 10 23-nucleotideaptamer 5′ terminus -G G rA rG rG [i2FU] [i2FG] C [i2FU] C C G A A A rGrG A A [i2FC] [i2FU] C C - 3′ terminus where A, C, G, U represent fournucleobases. Uppercase letter corresponds to deoxyribonucleotide, and rXindicates ribonucleotide. [i2FC] and [i2FU] denote 2′-fluoro substitutedpyrimidine nucleotides. 11 23-nucleotide aptamer 5′ terminus - G G rA rGrG [i2FU] rG rC [i2FU] C C G A A A rG rG A A [i2FC] [i2FU] C C - 3′terminus where A, C, G, U represent four nucleobases. Uppercase lettercorresponds to deoxyribonucleotide, and rX indicates ribonucleotide.[i2FC] and [i2FU] denote 2′-fluoro substituted pyrimidine nucleotides.12 23-nucleotide aptamer 5′ terminus - G G rA rG rG [i2FU] rG C rU C C GA A A rG rG A A [i2FC] [i2FU] C C - 3′ terminus where A, C, G, Urepresent four nucleobases. Uppercase letter corresponds todeoxyribonucleotide, and rX indicates ribonucleotide. [i2FC] and [i2FU]denote 2′-fluoro substituted pyrimidine nucleotides. 13 23-nucleotideaptamer 5′ terminus - G G rA rG rG [i2FU] rG C U(OMe) C C G A A A rG rGA A [i2FC] [i2FU] C C - 3′ terminus where A, C, G, U represent fournucleobases. Uppercase letter corresponds to deoxyribonucleotide, and rXindicates ribonucleotide. [i2FC] and [i2FU] denote 2′-fluoro substitutedpyrimidine nucleotides. U(OMe) represents 2′-methoxy substitutednucleotides.

We claim:
 1. A method of capturing human Fc domains found in a biofluidsample, the method comprising: providing an affinity capture devicecomprising a surface having an aptamer that binds to an Fc region;diluting the biofluid sample with a binding buffer comprising: (A)tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES),2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM; and adsorbing the human Fc domains in the biofluid sampleto the aptamer with the binding buffer.
 2. The method of claim 1,wherein the aptamer is a 23-nucleotide aptamer.
 3. The method of claim1, wherein the aptamer is non-covalently immobilized onto the surface ofthe affinity capture device.
 4. The method of claim 1, wherein theaptamer is covalently immobilized onto the surface of the affinitycapture device.
 5. The method of claim 1, wherein the binding buffer hasa concentration of monovalent cations less than about 50 mM.
 6. Themethod of claim 1, wherein the pH of the binding buffer is between about5 and about
 9. 7. The method of claim 1, wherein the biofluid sample isdiluted by a factor of 2, 10, or
 20. 8. The method of claim 1, whereinthe biofluid sample is diluted to obtain a total monovalent cationconcentation of the biofluid sample of no greater than 100 mM.
 9. Themethod of claim 1, further comprising eluting the adsorbed human Fcdomains from the immobilized aptamer using an eluent having an ammoniumconcentration between about 10 mM to about 1000 mM, wherein the ammoniumis in the form of tetramethylammonium, triethylammonium, ammoniumformate, or ammonium acetate.
 10. The method of claim 9, wherein theeluent has a pH between about 6.5 to about 8.0.
 11. The method of claim1, further comprising washing the adsorbed human Fc domains with thebinding buffer.
 12. The method of claim 1, further comprising washingthe adsorbed human Fc domains with a buffer comprising Ca⁺.
 13. Themethod of claim 1, wherein the concentration of the magnesium cation isbetween about 50 µM to about 1 mM.
 14. The method of claim 9, furthercomprising analyzing the eluted human Fc domains with a detector. 15.The method of claim 14, wherein the detector is a sandwiched enzymelinked immunosorbent assay or a mass spectrometer.
 16. A method ofcapturing human Fc domains found in a biofluid sample, the methodcomprising: providing an affinity capture device comprising a surfacehaving an anti-human Fc aptamer; diluting the biofluid sample with abinding buffer comprising: (A) tris(hydroxymethyl)aminomethane (Tris),trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM; and adsorbing the human Fc domains in the biofluid sampleto the aptamer with the binding buffer.
 17. A method of capturing humanFc domains found in a biofluid sample, the method comprising: providingan affinity capture device comprising a surface having a metal-dependentaptamer that binds to an Fc region; diluting the biofluid sample with abinding buffer comprising: (A) tris(hydroxymethyl)aminomethane (Tris),trimethylamine (TES), 2-ethanesulfonic acid (MES), or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) amagnesium cation at a concentration between about 10 µM to about 20 mM;and (C) a total monovalent cation concentration from 0 to no greaterthan 100 mM; and adsorbing the human Fc domains in the biofluid sampleto the aptamer with the binding buffer.